The present invention relates to the generation of excitation light in a system for detecting fluorescence emission, and more particularly to a control method for an apparatus for measuring fluorescence levels in samples.
The extent or rate of certain known chemical reactions can be measured by the alteration of levels of fluorescence when samples are illuminated by a suitable source of light. Likewise, some chemical and biological samples couple to fluorescent reporters such that measurement of fluorescence provides useful information regarding the makeup or quantity of the particular sample. Hence, devices for measuring and analyzing fluorescence are known in the art.
The basic process for illuminating and measuring fluorescence can be generally described as follows. A sample to be analyzed is placed in a suitable optical vessel such as a position or a well in a multiwell array. A source of light of suitable wavelength, such as an LED, a laser, or a lamp is directed toward the sample to excite the fluorescent molecules of the sample. The fluorescent molecules then emit light, which is read by an optical sensor. Certain characteristics of the emitted fluorescent light can be analyzed, and a scientific result obtained with regard to the nature or quantity of the particular sample.
An xe2x80x9cexcitation light sourcexe2x80x9d as utilized herein is intended to generally mean a source of light of a restricted wavelength range suitable for the excitation of fluorescence. Example excitation light sources include one or more light-emitting diodes, a combination of one or more light-emitting diodes and an optical filter, a combination of an incandescent light source and an optical filter, a combination of a fluorescent tube light source and an optical filter, and a laser such as a diode laser.
An xe2x80x9cemission light detectorxe2x80x9d as utilized herein is intended to generally mean a light sensor coupled with any necessary optical filters so as to be useful for detection of fluorescent emission, while being essentially insensitive to light at the excitation wavelength. This may include an amplifier circuit and other associated electronics.
The combination of conventional fluorometers with conventional thermal cyclers is known in the field of molecular biology. For instance, such instruments are described in U.S. Pat. Nos. 5,928,907, 6,015,674, 6,043,880, 6,144,448, 6,337,435, and 6,369,893. It is useful to create such combinations to perform different types of analysis. Some example applications and combinations can be found in U.S. Pat. Nos. 5,210,015, 5,994,056, 6,140,054, and 6,174,670. In typical procedures, fluorescence measurements are made during or after the Polymerase Chain Reaction (xe2x80x9cPCRxe2x80x9d) process to quantify or identify target DNA.
The invention is directed toward a particular class of fluorometers, disclosed in PCT Application Publication Number WO 01/35079 to E.I. DuPont De Nemours and Company, filed Nov. 9, 2000, which is hereby incorporated in its entirety by reference. FIG. 1 illustrates the elements that combine to form a basic fluorometer 10. The fluorometer 10 includes a supporting structure 12. A sample 14 for analysis resides in a position in the form of a well 16 of the fluorometer 10. The sample 14 is commonly in liquid form, but can be in other physical states. An excitation light source, such as one or more LEDs 18, directs excitation light to well 16 along approximately axis Axe2x80x94A. The excitation light illuminates the sample 14. The incident light excites fluorophores in the sample 14 and causes the fluorophores to fluoresce, emitting light of a relatively longer wavelength than the incident light. A portion of the emitted light is collected by an emission light detector, which can include a photomultiplier tube (PMT) 20. The PMT 20 converts the light into an electrical signal. For each sample well 16, there is a distinct excitation light source 18 so that multiple wells can be separately illuminated.
The amount of fluorescence emitting from a particular well 16 filled with the sample 14 is generally proportional to the amount of excitation light directed toward that well 16. In conventional fluorometers of similar design, the amount of excitation light reaching each well is not well controlled. More specifically, in the device described in PCT Application WO 01/35079, the amount of current driving each LED is substantially equal. However, differences in the efficiencies of individual LEDs result in different amounts of light output, and differences in the geometry of the excitation light path result in differences in the fraction of the excitation light that reaches each well. Furthermore, the efficiency of detection of fluorescence emission light varies depending on the geometry of the path from the sample well to the emission light detector.
The combination of these factors results in the need to calibrate each well by multiplying the fluorescence measurement of each well by a normalization factor. The result of such a correction is that the sensitivity and noise levels of each well vary as a function of well location, which can be undesirable. In addition, the accuracy of the resulting data is somewhat compromised, and small errors in measurement can be greatly amplified by multiplying the data by the normalization factors.